This protocol presents a comprehensive pipeline to analyze samples obtained from human hearts that span the microscopic and macroscopic scales.
Detailed study of non-failing human hearts rejected for transplantation provides a unique opportunity to perform structural analyses across microscopic and macroscopic scales. These techniques include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs) and immunohistochemical staining. Mesoscopic examination procedures include stereoscopic dissection and micro-computed tomographic (CT) scanning. Macroscopic examination procedures include gross dissection, photography (including anaglyphs and photogrammetry), CT, and 3D printing of the physically or virtually dissected or whole heart. Before macroscopic examination, pressure-perfusion fixation may be performed to maintain the 3D architecture and physiologically relevant morphology of the heart. The application of these techniques in combination to study the human heart is unique and crucial in understanding the relationship between distinct anatomic features such as coronary vasculature and myocardial innervation in the context of the 3D architecture of the heart. This protocol describes the methodologies in detail and includes representative results to illustrate progress in the research of human cardiac anatomy.
As function follows form, understanding the architecture of the heart is fundamental for appreciation of its physiology. Although numerous investigations have revealed cardiac anatomy from micro- to macroscales1,2,3, multiple questions remain unresolved, especially those related to human cardiac anatomy. This is in part because basic studies focusing on functional anatomy generally utilized animal hearts4,5,6, which are often distinct from human hearts1,7,8. Furthermore, each individual study, even those using human heart samples, tends to focus on very specific structures, which renders it difficult to apply the findings in the context of the whole heart. This is even more so if the focused structures are at micro- or mesoscales, such as the perinexus9 and ganglionated plexuses10.
In this context, systemic structural study of the human heart rejected for transplantation provides a unique and rare opportunity to obtain a comprehensive atlas of cardiac structures in focus across microscopic and macroscopic scales11. Microscopic examination protocols include tissue clearing (modified immunolabeling-enabled three-dimensional (3D) imaging of solvent-cleared organs, iDISCO+)12,13, and immunohistochemical staining. Mesoscopic examination protocols include stereoscopic dissection, macro photography, and micro-computed tomographic (CT) scanning. Macroscopic examination protocols include gross dissection14, photography (including anaglyphs and photogrammetry)15,16,17, CT, virtual dissection18, and 3D printing of the physically or virtually dissected or whole heart17. In preparation for macroscopic examination, pressure-perfusion fixation is performed to maintain the 3D architecture and physiologically relevant morphology of the heart14,19,20,21. The combined application of these techniques is unique and crucial to correlate distinct anatomic features in the context of the 3D architecture of the human heart.
As the opportunity to obtain a non-pathological human heart sample is extremely limited, a multi-scale approach described herein maximizes the use of the sample. By applying various procedures described below, representative results will illustrate to the reader how the findings can be utilized for multiple purposes, including discovery in scientific research11 (comprehensive analyses of cardiac innervation, distribution of ganglionated plexuses), improvement of clinical procedures (simulation for surgical and interventional approaches), and anatomical education (real 3D demonstration of cardiac anatomy).
This study used de-identified tissue samples collected from non-failing donor human hearts and was approved by the Institutional Review Board of the University of California, Los Angeles (UCLA). Samples were obtained from non-failing hearts that were rejected for transplantation. The hearts were pressure-perfused, fixed in 4% paraformaldehyde (PFA), and imaged before tissue processing per the following methods. Figure 1 summarizes the flow chart of the order of the study. The details of the reagents and the equipment used in the study are listed in the Table of Materials.
1. Micro-scale examination
2. Meso-scale examination
3. Macro-scale examination
Microscale examinations
Applying tissue clearing allows imaging of larger volumes of tissue in 3D using confocal microscopy. In the heart, ganglia containing cardiac neurons and the neural patterning of myocardial innervation can be visualized (Figure 2). Figure 3 shows a confocal image of the human left ventricle myocardium immunostained for nerves and smooth muscle cells. Blood vessels are noted to traverse the myocardium, and numerous nerve fibers are identified, both in association with and independent of blood vessels.
Meso- and macroscale examinations
When using absolute ethanol for 24 h pressure perfusion and fixation, the natural color of the sample is bleached, the tissue is dehydrated25, and elasticity is significantly reduced. On the other hand, upon fixation with PFA and formalin, natural color and elasticity are remarkably maintained. For these reasons, PFA or formalin is mainly used as the preferred fixative.
Figure 6 shows representative images of the gross dissection, virtual dissection, STL polygon model, and three-dimensional printing. Figure 7 shows representative images of the anaglyphs created from both gross and virtual dissection images. Depth perception can be obtained with anaglyphic glasses. The single photogrammetric model captured can be observed from almost all directions using commercially available software and demonstrates complex anatomical features relevant to routine transcatheter cardiac procedures. By applying these techniques to the heart prepared with pressure perfusion and fixation, three-dimensional information about the heart can be almost eternally retained either digitally or physically and shared without boundaries. Figure 8 shows the 50%-scale3D printings replicated from the dissected hearts rejected for transplantation.
Figure 1: Flow chart of the protocol. Please click here to view a larger version of this figure.
Figure 2: Tissue-cleared section of the human right atrium. (A) Right posterior oblique view of a human right atrium with the right atrial ganglionated plexus (RAGP) dissected for tissue clearing. (B) RAGP specimen before and after tissue clearing. (C) Maximum intensity projection of an iDISCO+-cleared portion of human RAGP demonstrating ganglia (arrowheads) immunostained with pan-neuronal marker protein gene product 9.5 (PGP9.5). Scale bars are 1 cm (A), 5 mm (B), and 500 µm (C). This figure is adapted from Hanna et al.11. Please click here to view a larger version of this figure.
Figure 3: Immunostained slides of the human left ventricle. Confocal image from the human left ventricle myocardium slice immunostained with pan-neuronal marker protein gene product 9.5 (PGP9.5) and smooth muscle cell marker α-smooth muscle actin (αSMA). Muscle autofluorescence is visible using the 488 nm laser line (green). The scale bar is 50 µm. Please click here to view a larger version of this figure.
Figure 4: Micro-computed tomographic imaging of heart samples. (A) Micro-computed tomography setup for heart sample imaging. (B) User interface for micro-computed tomography imaging. Please click here to view a larger version of this figure.
Figure 5: Photo-studio setting in UCLA Cardiac Arrhythmia Center. Please click here to view a larger version of this figure.
Figure 6: Gross dissection (upper left), virtual dissection (upper right), STL polygon model (bottom left), and three-dimensional printing (bottom right) images of the aortic and mitral valvular complex. Please click here to view a larger version of this figure.
Figure 7: Anaglyphs of a gross dissection (left) and virtual dissection (right) of the aortic and mitral valvular complex. Anaglyphic glasses (red/cyan) are necessary to obtain depth perception. Please click here to view a larger version of this figure.
Figure 8: Three-dimensional printing of heart samples. (A) Three-dimensional (3D) printer setup for heart sample 3D printing with a TPU filament. (B) Representative heart 3D prints produced using methods described in this study. Please click here to view a larger version of this figure.
The present study demonstrates the comprehensive pipeline to analyze samples obtained from whole human hearts. Representative results show micro- to macroscale anatomical examinations carried out routinely for a single heart. As a human heart sample is extremely precious, a multi-scale approach is ideal and effective so as not to waste any parts of the sample by applying multiple protocols for various purposes, including discovery in scientific research, improvement of clinical procedures, and anatomical education with maintaining anatomic correlation in the context of the whole heart.
Regarding microscale examination, immunostaining and microscopy may be applied to understand the cytoarchitecture of human cardiac specimens. Here, the application of tissue clearing and immunohistochemistry to study cardiac neuroanatomy at the cellular scale is demonstrated. The use of these techniques is helpful in the characterization of the cardiac nervous system and myocardial innervation patterns in relation to structures of interest, such as the cardiac conduction system and cardiac chambers. Although excellent spatial resolution is achieved, the use of confocal microscopy, particularly for tissue-cleared specimens, is time-consuming. Lightsheet microscopy may be used to reduce the time for image acquisition at the expense of spatial resolution.
Regarding meso- to macroscale examination, the spatial resolution of micro-CT scanners in the authors' institution ranges from 10-200 µm. The sample size is limited to 20 mm for a 10 µm scan, and 120 mm for a 100-200 µm scan. Micro-CT scanners in the authors' institution cannot accommodate the whole heart. Thus, at the authors' institution, a whole heart scan requires the use of a clinical CT scanner with 600 µm spatial resolution, although advances have allowed for the development of micro-CT scanners that can image the whole heart2. Technological development, such as photon counting CT, will surely expand further possibilities. Improvement of spatial resolution of the STL file should be the first step to further improve the quality of 3D printing. The higher cost of 3D printing limits the application of the technique to routine clinical practice. Photogrammetry images generated from any smartphone application are easy to develop and of acceptable quality but will require further sophisticated but expensive and time-consuming systems to improve resolution26. To visualize in 3D, extended reality with dedicated head gear27,28 and holographic monitors29 are additional tools but are also limited by higher cost.
In summary, through comprehensive structural analyses across microscopic and macroscopic scales, the microscale anatomy of each structure and its functional contribution can be understood in the context of the whole heart. Along with the development of high-resolution imaging, the distance between the micro- and macroscale anatomy is dramatically expanding. Experts in electron microscopic analysis of cardiomyocytes may not be familiar with the number of mitral leaflets and vice versa. To facilitate the comprehensive understanding of cardiac morphology, scientists must keep exploring further details of each tree, while maintaining the bird's eye view of the entire forest.
The authors have nothing to disclose.
We thank the individuals who have donated their bodies for the advancement of education and research. We are grateful to the OneLegacy Foundation, which formed the basis for obtaining donor hearts for research. We are also grateful to Anthony A. Smithson and Arvin Roque-Verdeflor of the UCLA Translational Research Imaging Center (Department of Radiology) for their support in CT data acquisition. This project was supported by the UCLA Amara Yad Project. We are thankful to Drs. Kalyanam Shivkumar and Olujimi A. Ajijola for establishing and maintaining a human heart pipeline for research. We appreciate our Research Operations Manager, Amiksha S. Gandhi for her dedication to support our projects. This work was made possible by support from NIH grants OT2OD023848 & P01 HL164311 and Leducq grant 23CVD04 to Kalyanam Shivkumar, the American Heart Association Career Development Award 23CDA1039446 to PH, and the UCLA Amara-Yad Project (https://www.uclahealth.org/medical-services/heart/arrhythmia/about-us/amara-yad-project). The GNEXT microPET/CT scanner used in this study was funded by an NIH Shared Instrumentation for Animal Research Grant (1 S10 OD026917-01A1).
1x Phosphate buffered saline | Sigma-Aldrich | P3813 | |
3D Viewer | Microsoft | ||
647 AffiniPure Donkey Anti-Rabbit IgG | Jackson ImmunoResearch Laboratories | 711-605-152 | |
647 AffiniPure Donkey Anti-Sheep IgG | Jackson ImmunoResearch Laboratories | 713-605-147 | |
AF Micro-NIKKOR 200 mm f/4D IF-ED lens | Nikon | ||
Anti-Actin, α-Smooth Muscle – Cy3 antibody | Sigma-Aldrich | C6198 | |
Antigen Retrieval Buffer (100x EDTA Buffer, pH 8.0) | Abcam | ab93680 | |
Anti-PGP9.5 (protein gene product 9.5) | Abcam | ab108986 | |
Anti-TH (tyrosine hydrox ylase) | Abcam | ab1542 | |
Anti-VAChT (vesicular acetylcholine transporter) | Synaptic Systems | 139 103 | |
Benzyl ether | Sigma-Aldrich | 108014 | |
Bovine serum albumin | Sigma-Aldrich | A4503-10G | |
Cheetah 3D printer filament (95A), 1.75 mm | NinjaTek | ||
Coverslip, 22 mm x 30mm, No. 1.5 | VWR | 48393 151 | |
Cy3 AffiniPure Donkey Anti-Rabbit IgG | Jackson ImmunoResearch Laboratories | 711-165-152 | |
Dichloromethane | Sigma-Aldrich | 270997-100ML | |
Dimethyl sulfoxide | Sigma-Aldrich | D8418-500ML | |
Ethanol, 100% | Decon laboratories | 2701 | |
Glycine | Sigma-Aldrich | G7126-500G | |
GNEXT PET/CT | SOFIE Biosciences | ||
Heparin sodium salt from porcine intestinal mucosa | Sigma-Aldrich | H3149-50KU | |
Histodenz | Sigma-Aldrich | D2158-100G | |
Hydrogen peroxide solution | Sigma-Aldrich | H1009-500ML | |
Imaging software | Zeiss | ZEN (black edition) | |
Imaging software | Oxford Instruments | Imaris 10 | |
iSpacer | Sunjin Labs | iSpacer 3mm | |
KIRI Engine | KIRI Innovation | ||
Laser scanning confocal microscope | Zeiss | LSM 880 | |
LEAD-2 – Vertical & Multi-channels Peristaltic Pump | LONGER | ||
Lightview XL | Brightech | ||
Methanol (Certified ACS) | Fischer Scientific | A412-4 | |
Nikon D850 | Nikon | ||
NinjaTek NinjaFlex TPU @MK4 | NinjaTek | ||
Normal donkey serum | Jackson ImmunoResearch Laboratories | 017-000-121 | |
Original Prusa MK4 3D printer | Prusa Research | ||
PAP pen | Abcam | ab2601 | |
Paraformaldehyde, 32% | Electron Microscopy Sciences | 15714-S | |
Polycam | Polycam | ||
Primary antibody | |||
PrusaSlicer 2.7.1 | Prusa Research | ||
SARA-Engine | pita4 mobile LLC | ||
Scaniverse | Niantic | ||
Secondary antibody | |||
SlowFade Gold Antiface Mountant | Invitrogen | S36936 | |
Sodium azide, 5% (w/v) | Ricca Chemical Company | 7144.8-32 | |
SOMATOM Definition AS | Siemens Healthcare | ||
Standard Field Surgi-Spec Telescopes, | Designs for Vision | ||
Stereomicroscope System SZ61 | OLYMPUS | ||
StereoPhoto Maker | Free ware developed by Masuji Suto | ||
Superfrost Plus Microscope Slides, Precleaned | Fisher Scientific | 12-550-15 | |
Triton X-100 | Sigma-Aldrich | T8787-50ML | |
Tween-20 | Sigma-Aldrich | P9416-100ML | |
Xylene | Sigma-Aldrich | 534056-4L | |
Ziostation2 | Ziosoft, AMIN |
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